The herpesvirus virion is comprised of four structural elements: a large double-stranded DNA molecule in the central core; an icosahedral capsid, which encloses the genome; a layer that immediately surrounds the capsid termed the tegument; and an outer membrane or envelope, which encloses the whole structure and in which are embedded the viral glycoproteins. The assembly and maturation of the infectious particle is a complex and poorly characterized process. The goal of this project is to elucidate the process of virion assembly in herpesviruses using a newly developed protein chip approach. Our working hypothesis is that virus assembly and maturation is a sequential process and is dependent on ordered interactions between multi-protein complexes. We will focus on analyzing protein interactions among 23 conserved structural proteins from each of alphaherpesvirus (HSV-1), betaherpesvirus (HCMV and HHV-7), and gammaherpesvirus (KSHV and EBV) families as well as two additional proteins specific for HSV-1, a total of 117 structural proteins. We will construct herpesvirus protein chips containing the 117 proteins and apply an innovative approach for identifying protein interactions at multiple levels. Further, we plan to identify the specific substrates of the conserved herpesvirus-encoded protein kinase (UL13, ORF36, BGLF4, UL97, and U69) as well as those of the alphaherpesvirus conserved protein kinase US3 using the above protein chips, and determine to what extent the phosphorylation of the identified substrates will affect their abilities to interact with other proteins/protein complexes. The integration of all the data will allow us to build a complex protein interaction network that will provide insights into the sequence and order of the virion assembly in herpesviruses. Herpesviruses are major human pathogens that cause life-long persistent infections and result in clinical manifestations that range from a mild cold sore, to ocular keratitis and even cancer. Thus, infections due to these viruses are a major public health concern and understanding the biology of herpesviruses is important in the development of efficacious treatments of these infections. The major practical significance that will result from the above studies is the identification of essential interactions that can be used to develop virus- specific antivirals. [unreadable] [unreadable] [unreadable]